Small molecule inhibition of microRNA-21 expression reduces cell viability and microtumor formation

Article abstract of DOI:10.1016/j.bmc.2019.05.044

MicroRNAs (miRNAs) are short, non-coding RNA molecules estimated to regulate expression of a large number of protein-coding genes and are implicated in a variety of biological processes such as development, differentiation, proliferation, and cell surviva

Full text of DOI:10.1016/j.bmc.2019.05.044

Accepted Manuscript
Small Molecule Inhibition of MicroRNA-21 Expression Reduces Cell Viability
and Microtumor Formation
Nicholas Ankenbruck, Rohan Kumbhare, Yuta Naro, Meryl Thomas, Laura
Gardner, Cole Emanuelson, Alexander Deiters
PII:
S0968-0896(19)30047-1
https://doi.org/10.1016/j.bmc.2019.05.044
BMC 14931
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 February 2019
1 May 2019
28 May 2019
Please cite this article as: Ankenbruck, N., Kumbhare, R., Naro, Y., Thomas, M., Gardner, L., Emanuelson, C.,
Deiters, A., Small Molecule Inhibition of MicroRNA-21 Expression Reduces Cell Viability and Microtumor
Formation, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.05.044
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Small Molecule Inhibition of MicroRNA-21 Expression Reduces Cell
Viability and Microtumor Formation
Nicholas Ankenbruck‡, Rohan Kumbhare‡, Yuta Naro‡, Meryl Thomas, Laura Gardner, Cole
Emanuelson, and Alexander Deiters*
‡ The authors contributed equally.
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United
States, deiters@pitt.edu
ABSTRACT
MicroRNAs (miRNAs) are short, non-coding RNA molecules estimated to regulate expression of
a large number of protein-coding genes and are implicated in a variety of biological processes
such as development, differentiation, proliferation, and cell survival. Dysregulation of miRNAs
has been attributed to the onset and progression of various human diseases, including cancer.
MicroRNA-21 (miR-21), one of the most established oncogenic miRNAs, is found to be
upregulated in a wide range of cancers making it an attractive therapeutic target. Employment of
a luciferase-based live-cell reporter assay in a high-throughput screen of >300,000 small
molecules led to the discovery of a new class of ether-amide miR-21 inhibitors. Following a
structure-activity relationship study, an optimized lead molecule was found to inhibit miR-21
transcription. Furthermore, the inhibitor demonstrated cytotoxicity in a cervical cancer cell line
via induction of apoptosis and was capable of reducing microtumor formation in a long-term
clonogenic assay. Altogether, this work reports the discovery of a new small molecule inhibitor
of miR-21 and demonstrates its potential as an alternative approach in cancer therapy.
1. INTRODUCTION
1

MicroRNAs (miRNAs) are small, non-coding RNA molecules of 20-22 nucleotides in length that
regulate gene expression. In the canonical pathway, miRNAs repress translation by hybridizing
to a complementary sequence in the 3’ untranslated region (3’ UTR) of target mRNAs.1 In
addition to inhibition of translation initiation factor binding and recruitment of deadenylases,
miRNAs have been shown to inhibit ribosome-mediated elongation and to displace translation
initiation factors post-translation initiation, ultimately resulting in mRNA decay. Recently,
miRNAs have also been predicted to repress translation via binding to the 5’ UTR, coding
domain sequences, and open reading frames.2 While some experimental studies have provided
support to these hypotheses,3-5 the generality and extent to which these alternative mechanisms
contribute to miRNA-mediated translational repression remains unclear. MiRNA biogenesis
occurs via a dedicated maturation pathway,6 beginning with transcription of primary miRNAs
(pri-miRNAs) by RNA polymerase II or III. Pri-miRNAs are subsequently cleaved by Drosha,
producing a short hairpin known as precursor-miRNA (pre-miRNA)7 which is then exported from
the nucleus to the cytoplasm8 where it is further processed by Dicer to yield the mature miRNA
duplex. The guide strand of mature miRNA is subsequently loaded into the RNA-Induced
Silencing Complex (RISC), which then modulates gene expression via sequence-specific
binding to the target mRNA.9,10 Using photoactivatable ribonucleoside enhanced cross-linking
and immunoprecipitation (PAR-CLIP), it was shown that miRNAs regulate 21% of protein-coding
genes in humans.11 Given the numerous genes targeted by miRNAs, it’s not surprising that their
dysregulation contributes to the development of various human diseases,12-14 most notably
cancer.15-18
MicroRNA-21 (miR-21) is an oncogenic miRNA found to be upregulated in almost all cancers
and it plays a critical role in carcinogenesis via repression of several important tumor
suppressors.19 Furthermore, miR-21 has been implicated in modulating drug resistance via
inhibition of programmed cell death protein 4 (PDCD4), an important target of miR-21, and a
2

subsequent increase in cellular inhibitor of apoptosis protein 2 (c-IAP2) activity.20 Additionally,
miR-21-mediated silencing of phosphatase and tensin homolog (PTEN) can lead to
chemoresistance via activation of the Akt and ERK pathways in cancer cells.21 Due to its
implications in cancer, miR-21 has emerged as a potential target for therapeutic approaches,
including small molecules.22-28
Here, we report a new class of small molecules that selectively inhibit miR-21 and that were
discovered through a high-throughput screen using a previously developed cell-based reporter
assay.23 Through extensive structure-activity relationship (SAR) studies, we identified an analog
with improved activity compared to the initial hit compounds. Mode of action studies suggest
that the small molecule inhibits miR-21 at the transcriptional step of miRNA biogenesis.
Furthermore, pre-therapeutic evaluation of the compound showed that small molecule-mediated
inhibition of miR-21 led to a decrease in cervical cancer cell proliferation and microtumor
formation, as well as induction of apoptosis via capase-3/7 activation.
2. RESULTS AND DISCUSSION
Previously, we developed a luciferase-based live cell reporter assay for the discovery of small
molecule modulators of miR-21 function.23 Briefly, a binding site complementary to miR-21 was
inserted downstream of a firefly luciferase gene in the 3’ UTR, such that expression of the
reporter in the presence of endogenous miR-21 would lead to a reduction in luminescence (Fig.
1A). Inhibition of miR-21 function relieves translational repression of firefly luciferase and leads
to an increase in luminescence signal. The reporter construct was stably introduced into HeLa
cells via lentiviral transduction to afford a cell line amenable to the high-throughput screening of
333,519 compounds (PubChem AID 2289), which was carried out by the National Cancer
3

Institute. Filtering hits through secondary assays using a miR-30a reporter (PubChem AID
2507) and an in vitro firefly luciferase assay (PubChem AID 493175) yielded 58 potential
inhibitors that were not general inhibitors of the miRNA pathway and were not false positives
due to non-miRNA specific firefly luciferase activation.29 Compound 1 (PubChem CID
71233585; Fig. 1B) was selected for further investigation based on its promising activity and
synthetic accessibility.
Figure 1. A) Design of a luciferase-based live-cell reporter for high-throughput screening of small
molecule modulators of miR-21 function. miR-21 binds to a complementary sequence in the 3’ UTR of the
luciferase gene inhibiting translation and resulting in a decrease in luciferase expression. Presence of a
small molecule inhibitor of miR-21 transcription relieves translational repression of the reporter, resulting
in an increase in luciferase levels. B) Chemical structure of hit compound 1 identified in the high-
throughput screen.
Analogs of the initial hit 1 were synthesized using a synthetic route that allowed for facile
functionalization of numerous positions across the structure (Scheme S1). Commercially
available ortho-substituted anilines 2 were readily reacted with 3 via a condensation reaction in
polyphosphoric acid to obtain the resulting benzoimidazoles, benzothiazoles, and benzoxazoles
4. Subsequent reaction with acetyl chloride provided the linker region in 5 which readily
underwent nucleophilic substitution with phenols, thiols and anilines in the presence of cesium
carbonate to yield the final products.
Using this synthetic route, a small panel of analogs was generated and their biological activity
was evaluated using the HeLa-miR21-Luc cell line (Fig. 2). The initial hit 1 demonstrated
4

modest activity, inducing a 1.55-fold increase in luciferase signal following treatment at 10 µM
for 48 h. Removal of the iodide to yield 6 resulted in only a very minor improvement in activity,
while loss of the iodide along with introduction of a para-methoxy group in 7 had little to no
effect on activity. Turning towards modifying the benzothiazole ring, the benzoxazole derivative
of 1 yielded 8, which maintained the same activity as the parent compound. Keeping the
benzoxazole and removal of the iodide resulted in 9, which showed no change in activity, while
introduction of a para-methoxy to yield 10 abolished all activity. Expanding upon 9, addition of a
5-chloro modification to the benzoxazole led to in 11, which demonstrated an 85%
enhancement in activity compared to the initial hit 1. Furthermore, addition of a para-methoxy
substitution to 11 to yield 12 further enhanced activity, eliciting a 314% improvement compared
to 1. Continuing from the promising results obtained from 11 and 12, analogs containing
benzimidazoles (13 and 14) were synthesized, however, only a loss in activity was observed in
both cases.
O
O
O
O
O
O
O
O
O
N
S
N
S
N
S
N
O
NH
NH
I
NH
NH
I
7
8
6
1
1.56 ± 0.20
1.53 ± 0.30
1.68 ± 0.04
1.55 ± 0.19
O
O
O
O
O
O
O
O
Cl
Cl
N
N
O
O
O
N
O
NH
NH
NH
N
O
NH
10
O
11
12
9
0.27 ± 0.14
2.87 ± 0.31
4.87 ± 0.22
1.47 ± 0.11
O
O
O
O
O
Cl
N
Cl
N
NH
NH
N
N
H
H
14
13
0.41 ± 0.14
1.60 ± 0.32
Figure 2. Activity of compounds 1 and 6 – 14 in the HeLa-miR21-Luc assay. Values represent fold-
changes in luminescence signal in the HeLa-miR21-Luc stable cell line normalized to cell viability and
relative to a DMSO control. Data represents the average ± standard deviation from at least three
independent experiments.
To build upon the improvement in activity seen in 11 and 12, an additional round of analogs was
generated containing further modification to each respective compound. For 11, moving the 5-
5

chloride to the 6-position yielded 16, which had only a minor impact on activity. Changing the
ether-amide linker from the para- to the meta-position yielded analogs 17 and 18, which resulted
in a loss of activity. The importance of the ether-amide linker was investigated by synthesizing
19 bearing a shorter carbamate linker (Scheme S2). Compound 19 elicited a 35% increase in
activity relative to 11 and a 249% increase relative to the initial hit 1. Analogous modifications
were also made to 12 to produce 20, 21, 22, and 23, however, only reduced activity was
observed in this set of analogs (Fig. 3).
O
HN
O
O
Cl
O
O
O
N
O
H
N
O
Cl
N
O
NH
NH
Cl
17
16
11
2.14 ± 0.32
2.65 ± 0.26
2.87 ± 0.31
O
O
HN
Cl
H
O
O
N
N
NH
O
O
19
18
3.86 ± 0.64
1.75 ± 0.36
O
HN
Cl
O
O
O
O
N
O
O
O
O
H
N
Cl
N
O
NH
NH
O
Cl
21
O
20
12
3.19 ± 0.57
O
3.32 ± 0.07
4.87 ± 0.22
O
O
HN
Cl
H
O
O
N
N
O
NH
O
23
22
3.48 ± 0.48
O
4.73 ± 1.05
Figure 3. Activity of compounds 16 – 23 in the HeLa-miR21-Luc assay. Values represent fold-changes in
luminescence signal in the HeLa-miR21-Luc stable cell line normalized to cell viability and relative to a
DMSO control. Data represents the average ± standard deviation from at least three independent
experiments.
With 12 remaining the most potent inhibitor identified thus far, we decided to investigate further
modifications to its structure. To this end, analogs containing modifications to the ether-amide
linker and the central aniline ring were synthesized and their activity was tested in the HeLa-
miR21-Luc stable cell line (Fig. 4). Replacement of the ether-amide linker with a thioether amide
6

linker produced analog 24 which exhibited a 54% decrease in activity relative to 12.
Furthermore, incorporation of an amino-amide linker in place of the ether-amide linker resulted
in 25 and completely abolished activity. Replacement of the central aniline ring with a
naphthalene (26) or benzothiazole (27) was also detrimental to activity. Lastly, alkylation of the
amide nitrogen in 12 with a methyl or propargyl group, 28 and 29 respectively (Scheme S3),
abolished activity compared to the parent compound 12. Taken together, these results
confirmed the importance of the ether-amide linker to activity and thus we decided to maintain
this moiety moving forward with additional analogs.
O
O
O
O
S
O
O
HN
O
Cl
N
O
Cl
Cl
N
O
N
O
NH
NH
NH
24
12
4.87 ± 0.22
25
2.64 ± 0.41
0.14 ± 0.404
O
H
O
N
O
O
O
O
O
N
S
Cl
N
O
Cl
N
Cl
NH
N
O
N
O
O
O
O
28
27
26
1.30 ± 0.35
0.88 ± 0.84
0.03 ± 0.01
O
N
O
Cl
N
O
29
1.61 ± 0.10
Figure 4. Activity of compounds 24 – 29 in the HeLa-miR21-Luc assay. Values represent fold-changes in
luminescence signal in the HeLa-miR21-Luc stable cell line normalized to cell viability and relative to a
DMSO control. Data represents the average ± standard deviation from at least three independent
experiments.
Building upon the generated structure-activity information, we decided to synthesize an
additional set of analogs based on 12, but containing more diverse changes to the two distal
ring systems while maintaining the structure of the central region of the scaffold. To this end,
additional analogs were synthesized and tested in the miR-21 luciferase reporter (Fig. 5).
Moving the para-methoxy to the meta-position resulted in 30, which displayed similar activity to
12. Replacement of the para-methoxy with a nitrile (31), fluorine (32), nitro (33), or phenyl (34)
7

group all resulted in losses of activity. Similarly, introduction of a para-isopropyl in conjunction
with a meta-methyl group (35) led to a 46% decrease in activity. Interestingly, replacement of
the 5-chloro with a 5-fluoro in conjunction with (36) or without (37) the para-methoxy group also
reduced activity, providing evidence that the 5-position may be highly sensitive to modifications.
To further support this, introduction of a bromine at the 5-position (38) resulted in a 27%
increase in activity compared to the parent compound 12. Surprisingly, introduction of a phenyl
group at the 5-position (39) had an even greater impact on activity, demonstrating a 62%
improvement in activity compared to 12, and a 508% increase in activity relative to the initial hit
1. Conversion of the benzoxazole to a pyridinyloxazole (40) led to a loss in activity compared to
1 and further supports the critical nature of the 5-position on the benzoxazole.
O
O
O
O
O
CN
O
O
O
O
F
Cl
N
O
Cl
Cl
N
O
Cl
N
O
N
O
NH
NH
NH
O
NH
12
31
32
30
5.37 ± 1.20
4.87 ± 0.22
2.08 ± 0.20
2.86 ± 0.14
O
O
O
O
O
O
O
O
O
NO2
O
O
F
N
O
Cl
Cl
N
O
N
O
Cl
N
NH
NH
NH
NH
O
36
35
33
34
2.64 ± 0.20
2.14 ± 0.54
2.23 ± 0.50
3.58 ± 0.11
O
O
O
O
O
O
O
O
O
N
N
O
F
N
O
N
O
Br
N
NH
NH
NH
NH
O
40
39
37
38
6.19 ± 0.78
2.25 ± 0.23
7.87 ± 0.24
1.58 ± 0.18
Figure 5. Activity of compounds 30 – 40 in the HeLa-miR21-Luc assay. Values represent fold-changes in
luminescence signal in the HeLa-miR21-Luc stable cell line normalized to cell viability and relative to a
DMSO control. Data represents the average ± standard deviation from at least three independent
experiments.
Overall, the SAR studies suggest several key positions on the inhibitor that appear sensitive to
modification, including the ether-amide linker core and the 5-position on the benzoxazole ring
system. With the promising and improved candidates 12, 30, 38, and 39 in hand, we next
evaluated their biological activity and confirmed their function as miR-21 inhibitors.
8

2.2. Secondary assays to confirm activity of miR-21 inhibitors
Firstly, the HeLa-miR21-Luc reporter cell line was treated in dose-response with the most
promising analogs, and 12, 30, 38, and 39 all demonstrated reporter activation in a dose-
dependent fashion with EC50 values of 6.7 µM, 4.7 µM, 6.4 µM, and 3.5 µM, respectively (Fig.
6).
12
30
38
39
EC50 = 6.7 µM
EC50 = 4.7 µM
EC50 = 6.4 µM
EC50 = 3.5 µM
10
8
10
8
10
8
10
8
6
6
6
6
4
4
4
4
2
2
2
2
0
0
0
0
-0.5 0.0
0.5
1.0
1.5
2.0
-0.5 0.0
0.5
1.0
1.5
2.0
-0.5 0.0
0.5
1.0
1.5
2.0
-0.5 0.0
0.5
1.0
1.5
2.0
concentration / log[µM]
concentration / log[µM]
concentration / log[µM]
concentration / log[µM]
Figure 6. Treatment of HeLa-miR21-Luc stable reporter cells with varying concentrations of inhibitor 12,
30, 38, or 39 followed by a Bright-Glo assay. Luciferase signal was first normalized to cell viability and
then to a DMSO control. Errors bars represent standard deviations from three independent experiments.
EC50 values were determined by fitting data to a nonlinear regression analysis in GraphPad Prism
software.
Firefly luciferase inhibitors have been previously identified as false positive hits in high-
throughput screens due to luciferase enzyme stabilization in cells.30 Because this is a
documented concern when using luciferase reporter assays,29 we decided to evaluate the effect
of 12, 30, 38, and 39 on luciferase activity in a biochemical assay. Treatment with the positive
control firefly luciferase inhibitor PTC-12419 led to a 94% reduction in enzyme activity. While
treatment with 12 elicited no significant reduction in luciferase activity, treatment with 30, 38,
and 39 all led to 44, 38, and 27% reductions in luminescence signal, respectively (Fig. 7A). To
further validate whether these inhibitors act on the miRNA pathway, we transfected HeLa cells
with a psiCHECK-empty reporter (containing a sequence not targeted by any known miRNA)
followed by treatment with the compounds at 10 µM for 48 h. The positive control PTC-124 and
9

compounds 30, 38, and 39 induced an increase in luminescence signal ranging from 1.5 to 1.8-
fold, while compound 12 only showed a minor increase in luciferase activity (Fig. 7B). These
biochemical and cell-based results indicate that 30, 38, and 39 may have inflated activity
profiles in the HeLa-miR21-Luc assay due to off-target binding to the firefly luciferase enzyme.
Taking both the dose-response and in vitro firefly luciferase data into account, we decided to
carry 12 forward as the primary candidate for further biological testing.
Figure 7. Evaluation of selectivity of lead compounds. A) Incubation of recombinant firefly luciferase with
inhibitors 12, 30, 38, or 39 at 10 µM, followed by a Bright-Glo assay. PTC-124, a known firefly luciferase
inhibitor, was included as a positive control. B) Transfection of HeLa cells with the psiCHECK-empty
plasmid followed by treatment with compounds 12, 30, 38, and 39 or the positive control at 10 µM. A dual
luciferase assay was performed after 48 h. RLU values represent firefly luciferase luminescence signal
normalized to Renilla luciferase luminescence signal. C) Treatment of Huh7-miR122 stable cells with 12
(10 µM) or positive control 41 (10 µM). A dual luciferase assay was performed after 48 h. RLU values
represent Renilla luciferase luminescence signal normalized to firefly luciferase luminescence signal. All
data were normalized to DMSO treatment (negative control) and errors bars represent standard
deviations from three independent experiments. Statistical significance was determined using an unpaired
t-test, * P < 0.05, ns P ≥ 0.05.
Next, we tested if 12 is a general miRNA pathway inhibitor or if it has selectivity for miR-21. To
this end, we employed an additional stable reporter cell line, Huh7-miR122, which places
Renilla luciferase expression under the control of miR-122 activity.31 Treatment with 12 (10 µM)
elicited only a minor 0.2-fold reduction in Renilla luciferase signal, demonstrating that 12 does
not inhibit miR-122 activity (Fig. 7C). As a positive control, cells were also treated with 41, a
known miR-122 inhibitor,31 which induced a >3.5-fold increase in luminescence, confirming
functionality of the assay. Taken together, these results indicate that 12 shows some level of
selectivity for miR-21 and is not a general miRNA pathway inhibitor.
10

2.3. Identification of preliminary mode of action
To explore the mechanism by which 12 inhibits miR-21 function, levels of mature miR-21 were
measured via reverse transcription quantitative PCR (RT-qPCR) after treatment with 12. To
ensure that the effects were not cell line dependent, three different cancer cell lines were tested.
These cell lines included the parental line of the HeLa-miR21-Luc stable reporter cell line, HeLa
cervical carcinoma cells, along with A549 non-small cell lung carcinoma cells, and SKOV3
ovarian cancer cells. Each cell line was treated with 12 (10 µM) or DMSO (0.1%) for 48 h,
followed by miRNA isolation and quantification using RT-qPCR. In HeLa cells, a 57% reduction
was observed (Fig. 8A), while in A549 and SKOV3 cells, 33% and 31% reductions were
detected, respectively (Fig. S1). These results support a mechanism by which treatment with 12
results in depletion of mature miR-21 and subsequent reduction of miR-21 regulatory functions.
To determine if 12 inhibits miR-21 maturation via binding directly to pre-miR-21, melting curve
analyses were carried out in the presence of DMSO or 12. The melting temperature of pre-miR-
21 in the presence of DMSO control was measured to be 56.0 °C. The presence of 12 (10 µM)
had no effect on melting temperature yielding a Tm of 56.2 °C (Fig. S2). These results indicate
that 12 does not directly interact with miR-21 RNA and that miR-21 levels are likely impacted via
an alternative mechanism.
Figure 8. A) Treatment of HeLa cells with 12 (10 µM) for 48 h, followed by RT-qPCR analysis of mature
miR-21 or primary miR-21 (pri-miR-21). B) Transfection of HeLa cells with a miR-21 promoter gene
expression reporter followed by treatment with 12 (10 µM). A Bright-Glo assay was performed after 48 h.
11

Data was normalized to DMSO treatment (negative control) and errors bars represent standard deviations
from three independent experiments. Statistical significance was determined using an unpaired t-test, * P
< 0.05.
To further investigate what step of the miR-21 biogenesis pathway our inhibitor may be
targeting, primary miR-21 levels were analyzed via RT-qPCR in HeLa cells after treatment with
12 (10 µM) for 48 h. Interestingly, a marked 79% decrease in primary miR-21 levels was
observed following treatment (Fig. 8A). This result supports a mechanism by which 12 is
inhibiting transcription of the miR-21 gene, resulting in depletion of primary miR-21 levels. To
confirm that compound 12 inhibits miR-21 transcription, HeLa cells were transfected with a
previously described luciferase reporter construct that monitors miR-21 promoter activity, pGL4-
miR21P,22 and were treated with DMSO or 12 (10 µM) for 48 h. As expected, treatment with 12
resulted in a 78% decrease in miR-21 promoter activity compared to DMSO control (Fig. 8B).
This result directly supports the drastic reduction in primary miR-21 levels observed via RT-
qPCR and further suggests a mechanism of action by which 12 inhibits transcription of the miR-
21 gene.
2.4. Functional studies of 12
Next, we evaluated whether 12-mediated inhibition of miR-21 could elicit a therapeutic response
in HeLa cells by subjecting them to a cell viability study using an XTT assay.32 Following
treatment with the compound for 72 h, 12 demonstrated a disappointing IC50 > 50 µM (Fig. 9A).
Additionally, treatment of HEK293T cells, which do not express miR-21, with a serial dilution of
compound 12 did not inhibit cell viability, supporting our hypothesis that the compound reduces
HeLa cell viability in a miR-21-dependent manner (Fig. S3). To further explore this response
and determine if 12-induced limited cell death was due to triggering of apoptosis, we examined
caspase-3/7 activation in HeLa cells. Antisense oligonucleotide-mediated inhibition of miR-21
has been previously shown to inhibit cell proliferation and increase apoptosis of HeLa cells.33
12

Treatment of HeLa cells with 12 (10 M) for 24 h elicited a modest 50% increase in caspase-3/7
activity, a 2-fold increase was observed at an increased concentration of 50 M (Fig. 9B). This
suggests that knockdown of miR-21 by 12 leads to a reduction in cell viability by inducing
apoptosis, consistent with previous reports.33-36
We further explored the therapeutic potential of 12 by investigating the ability of the small
molecule to elicit long-term effects in reducing microtumor formation in a clonogenic assay.
Oligonucleotide silencing of miR-21 has been shown to inhibit clonogenicity in several cancer
cell models37-39 including cervical cancer.33,40 Briefly, HeLa cells were suspended in low melt
agarose and exposed to a dilution series of 12 for two weeks at 37 ºC prior to imaging (Fig. S4).
Interestingly, while treatment with 12 only inhibited cell viability at high concentrations, the
compound was capable of reducing colony formation of HeLa cells with an EC50 of 7.3 µM (Fig.
9C). This inhibition of microtumor formation is in good agreement with previous reports that
have shown similar results using antisense oligonucleotide-mediated knockdown of miR-
21.33,37,41,42 Furthermore, it was also reported that inhibition or knockout of miR-21 with genetic
tools had limited or no effect on cell viability, depending on the cell line,43,44 while connections
between miR-21 and metastasis and tumorigenesis have been observed in a variety of
cancers.45-48
Figure 9. Pre-therapeutic evaluation of 12 in HeLa cells A) Treatment of HeLa cells with 12 for 72 h,
followed by a cell viability assay. B) Treatment of HeLa cells with 12 in a two-week clonogenic assay. C)
Treatment of HeLa cells with 12 for 24 h, followed by a caspase-3/7 activity assay. Data was normalized
to DMSO treatment (negative control) and errors bars represent standard deviations from three
independent experiments.
13

3. SUMMARY
In summary, a high-throughput screen for small molecule modulators of miR-21 function led to
the identification of a new ether-amide scaffold, and a subsequent structure-activity relationship
study of the initial hit compound 1 resulted in the identification of the improved miR-21 inhibitor
12. Through a biochemical and cell-based firefly luciferase assays as well as a miR-122 reporter
cell line, we determined that compound 12 showed selectivity for inhibiting miR-21 function.
Furthermore, 12 was found to inhibit transcription of the miR-21 gene resulting in significant
reductions in primary and mature miR-21 levels. This mechanism of action is in contrast to other
classes of miR-21 inhibitors which appear to inhibit maturation of pre-miR-2122,25-27 as well as
oxadiazole-based inhibitors which appear to inhibit the function of mature miR-21 without
affecting miR-21 levels.24 However, the ether-amide class does appear to follow a similar
mechanism to a previously reported azobenzene-based miR-21 inhibitor.23 Pre-therapeutic
evaluation of the lead compound using toxicity and caspase activation assays showed that miR-
21 inhibition mediated by 12 reduces cell viability in HeLa cells by inducing apoptosis, although
high concentrations are required. However, treatment of HeLa cells with 12 in a long-term
clonogenic assay effectively inhibited microtumor formation at low micromolar doses,
suggesting that the inhibitor 12 may have potential as a therapeutic for treating miR-21-related
diseases.
4. EXPERIMENTAL SECTION
4.1. Chemistry
All reactions were performed in flame-dried glassware under a nitrogen atmosphere and stirred
magnetically. Reactions were followed by thin layer chromatography (TLC) using glass-backed
silica gel plates (Sorbent Technologies, 250 μm thickness). Anhydrous acetonitrile,
tetrahydrofuran (THF) were purchased from Acros and anhydrous toluene, dimethylformamide
14

(DMF) were purchased from Alfa Aesar. Silica gel column chromatography was performed on
silica gel (60 Å, 40-63 μm, 230 × 400 mesh, Sorbtech) as a stationary phase. High resolution
mass spectral analysis (HRMS) was performed on a Q-Exactive (Thermo Scientific) mass
1
spectrometer. The H NMR and 13C NMR spectra were recorded on a 300 MHz or 400 MHz
Varian NMR spectrometers. Chemical shifts are given in δ units (ppm) for 1H NMR spectra and
13C NMR spectra.
4.1.1. General procedure for synthesis of compounds 1, 6-18, 20-22, 24-27, 30-40.
Polyphosphoric acid (10 g) was added to the mixture of 2-aminophenols (or 2-aminothiols or 2-
phenylenediamines) 2 (7 - 9 mmol, 1.0 eq) and carboxylic acids 3 (1.0 eq) at room temperature
and heated to 170 °C (dissolution of reactants in polyphosphoric acid and effective stirring was
observed at elevated temperatures). The reaction mixture was stirred at 170 °C for 3 h and was
then allowed to cool to room temperature. The viscous reaction mixture was slowly diluted by
ice-water (200 mL), neutralized (to pH 7) by saturated solution of NaHCO3 and extracted with
multiple portions of ethyl acetate (3 × 50 mL). The combined organic extracts were washed with
brine (50 mL), dried over sodium sulfate (30 g), filtered and concentrated to yield compounds 4
in 24-91% yields as colored solids.
Further, compounds 4 (0.35 - 0.50 mmol, 1.0 eq) were heated with chloroacetyl chloride (3.0 eq)
in anhydrous toluene (10 mL) to 90 °C for 2 h. This reaction mixture was concentrated under
reduced pressure and the residue was triturated with diethyl ether (10 mL). The product was
filtered and subsequently washed with multiple portions of diethyl ether (3 × 5 mL) and dried to
recover colored solids. The solids were vigorously stirred in a saturated solution of NaHCO3 (10
mL) for 30 min, acidified to neutral pH by aqueous 1 M HCl solution and then extracted with
multiple portions of ethyl acetate (5 × 10 mL) until the aqueous layer turns colorless. The
combined organic extracts were washed with brine (10 mL), dried over sodium sulfate (10 g),
were filtered and concentrated to yield pure amides 5 as colored solids in 70-90% yield.
15

To a solution of amides 5 (0.08 - 0.44 mmol, 1.0 eq) in anhydrous acetonitrile (5 mL), the
indicated phenols, thiophenols, or anilines (1.2 eq) and Cs2CO3 (1.2 eq) were added and the
reaction mixture was stirred overnight at room temperature. The reaction was quenched by
adding water (10 mL) and extracted with ethyl acetate (5 × 10 mL). The combined ethyl acetate
extracts were washed with brine (10 mL), dried over sodium sulfate (10 g), filtered and
concentrated under reduced pressure. The residual solid was purified by silica gel column
chromatography in 1:9 – 7:3 ethyl acetate/hexanes to yield compounds 1, 6-18, 20-22, 24-27,
30-40 in 23% to quantitative yield.
Compounds 6 (PubChem CID 871321) and 7 (PubChem CID 82391) have been previously
characterized.
1
N-(4-(Benzo[d]oxazol-2-yl)phenyl)-2-(2-iodophenoxy)acetamide (8). 58 mg, 39% yield; H
NMR (300 MHz, CHCl3-d) δ 9.07 (s, 1H), 8.28 (d, J = 8.8 Hz, 1H), 7.89 - 7.76 (m, 4H), 7.61 -
7.58 (m, 1H), 7.40 - 7.32 (m, 2H), 6.88 (t, J = 14.4 Hz, 2H), 6.83 - 6.76 (m, 2H), 4.69 (s, 2H).
HRMS (ESI) calcd. for C21H15N2O3I (M+H)+ 471.0206, found: 471.0226.
N-(4-(Benzo[d]oxazol-2-yl)phenyl)-2-phenoxyacetamide (9). 35 mg, 90% yield; 1H NMR (300
MHz, CHCl3-d) δ 8.47 (s, 1H), 8.26 (d, J = 8.7 Hz, 2H), 7.82 - 7.75 (m, 3H), 7.61 - 7.55 (m, 1H),
7.40 - 7.33 (m, 3H), 7.08 - 7.00 (dd, J = 15.6, 6.6 Hz, 3H), 4.66 (s, 2H). HRMS (ESI) calcd. for
C21H16N2O3 (M+H)+ 345.1239, found: 345.1269.
N-(4-(Benzo[d]oxazol-2-yl)phenyl)-2-(4-methoxyphenoxy)acetamide (10). 16 mg, 52% yield;
1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1 H), 8.26 (d, J = 8.4 Hz, 2 H), 7.76 - 7.80 (m, 3 H),
7.57 - 7.58 (m, 1 H), 7.34 - 7.36 (m, 2 H), 6.80 - 6.96 (m, 4 H), 4.59 (s, 2 H), 3.79 (s, 3H); HRMS
(ESI) calcd. for C22H17O4N2 (M+H)+ 373.1183, found: 373.1187.
16

N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-phenoxyacetamide (11). 21 mg, 56% yield; 1H
NMR (400 MHz, DMSO-d6) δ 8.47 (s, 1 H), 8.23 (d, J = 8.4 Hz, 2 H), 7.80 (d, J = 8.8 Hz, 2 H),
7.24 (d, J = 2.2 Hz, 1 H), 7.49 (d, J = 8.2 Hz, 1 H), 7.37 - 7.39 (m, 2 H), 7.32 - 7.33 (m, 1 H),
7.00 - 7.08, 3 H), 4.65 (s, 2 H); HRMS (ESI) calcd. for C21H16O3N2Cl (M+H)+ 379.0844, found:
379.0848.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(4-methoxyphenoxy)acetamide (12). 20 mg,
52% yield; 1H NMR (400 MHz, CHCl3-d) δ 8.48 (s, 1 H), 8.23 (d, J = 8.2 Hz, 2 H), 7.81 - 7.79 (m,
2 H), 7.72 (d, J = 2.2 Hz, 1 H), 7.50 (d, J = 8.8 Hz, 2 H), 7.30 - 7.33 (m, 1 H), 6.88 - 6.96 (m, 4
H), 4.60 (s, 2 H), 3.79 (s, 3H); 13C NMR (100 MHz, CHCl3-d) δ 166.73, 162.59, 155.14, 151.00,
150.74, 142.16, 139.80, 128.73, 125.03, 124.61, 123.37, 119.90, 119.86, 116.01, 115.02,
110.55, 68.56, 55.73; HRMS (ESI) calcd. for C22H16O4N2Cl (M+H)+ 407.0793, found: 407.0796.
N-(4-(5-Chloro-1H-benzo[d]imidazol-2-yl)phenyl)-2-(4-methoxyphenoxy)acetamide (13). 22
mg, 58% yield; 1H NMR (400 MHz, DMSO-d6) δ 13.01 (br s, 1 H), 10.29 (s, 1 H), 8.08 - 8.15 (m,
2 H), 7.80 - 7.87 (m, 2 H), 7.47 - 7.69 (m, 2 H), 7.21 (dd, J = 8.4, 1.8 Hz, 1 H), 6.85 - 7.01 (m, 4
H), 4.67 (s, 2 H), 3.70 (s, 3 H); HRMS (ESI) calcd. for C22H19O3N3Cl (M+H)+ 408.1109, found:
408.1120.
N-(4-(5-Chloro-1H-benzo[d]imidazol-2-yl)phenyl)-2-phenoxyacetamide (14). 21 mg, 60%
yield; 1H NMR (400 MHz, DMSO-d6) δ 13.01 (d, J = 12.4 Hz, 1 H), 10.35 (s, 1 H), 8.09 - 8.16 (m,
2 H), 7.80 - 7.87 (m, 2 H), 7.62 - 7.70 (m, 1 H), 7.50 - 7.54 (m, 1 H), 7.30 - 7.36 (m, 2 H), 7.21
(ddd, J = 9.9, 8.3, 2.0 Hz, 1 H), 6.96 - 7.04 (m, 3 H), 4.75 (s, 2 H); HRMS (ESI) calcd. for
C21H17O2N3Cl (M+H)+ 378.1004, found: 378.1007.
1
N-(4-(6-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-phenoxyacetamide (16). 12 mg, 32% yield; H
NMR (300 MHz, DMSO-d6) δ 10.41 (s, 1 H), 8.15 (d, J = 8.6 Hz, 2 H), 7.89 - 7.99 (m, 3 H), 7.79
17

(d, J = 8.5 Hz, 1 H), 7.45 (dd, J = 8.4, 2.0 Hz, 1 H), 6.86 - 7.01 (m, 4 H), 4.69 (s, 2 H), 3.70 (s, 3
H); HRMS (ESI) calcd. for C21H16O3N2Cl (M+H)+ 379.08440, found: 379.0862.
1
N-(3-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-phenoxyacetamide (17). 23 mg, 62% yield; H
NMR (300 MHz, DMSO-d6) δ ppm 8.35 - 8.52 (m, 1 H), 8.31 (s, 1 H), 7.90 - 8.03 (m, 2 H), 7.71
(d, J = 1.7 Hz, 1 H), 7.42 - 7.56 (m, 2 H), 7.22 - 7.38 (m, 3 H), 6.96 - 7.09 (m, 2 H), 4.63 (s, 2 H);
HRMS (ESI) calcd. for C21H14O3N2Cl (M+H)+ 377.0688, found: 377.0697.
N-(3-(Benzo[d]oxazol-2-yl)phenyl)-2-phenoxyacetamide (18). 38 mg, 97% yield; 1H NMR
(400 MHz, DMSO-d6) δ 10.41 (s, 1 H), 8.6 (t, J = 1.8 Hz, 1 H), 7.9 (dt, J = 7.6, 1.3 Hz, 1 H), 7.79
- 7.85 (m, 3 H), 7.67 - 7.79 (m, 1 H), 7.58 (t, J = 7.9 Hz, 1 H), 7.41 - 7.47 (m, 2 H), 7.30 - 7.37
(m, 2 H), 6.97 - 7.07 (m, 3 H), 4.76 (s, 2 H); HRMS (ESI) calcd. for C21H17O3N2 (M+H)+
345.1233, found: 345.1249.
N-(4-(6-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(4-methoxyphenoxy)acetamide (20). 25 mg,
65% yield; 1H NMR (300 MHz, DMSO-d6) δ 10.41 (s, 1 H), 8.15 (d, J = 8.6 Hz, 2 H), 7.89 - 7.99
(m, 3 H), 7.79 (d, J = 8.5 Hz, 1 H), 7.45 (dd, J = 8.4, 2.0 Hz, 1 H), 6.86 - 7.01 (m, 4 H), 4.69 (s, 2
H), 3.70 (s, 3 H); HRMS (ESI) calcd. for C22H18O4N2Cl (M+H)+ 409.0949, found: 409.0964.
N-(3-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(4-methoxyphenoxy)acetamide (21). 35 mg,
1
89% yield; H NMR (300 MHz, DMSO-d6) δ 10.36 (s, 1 H), 8.68 (s, 1 H), 7.81 - 7.98 (m, 4 H),
7.45 - 7.62 (m, 2 H), 6.86 - 7.03 (m, 4 H), 4.68 (s, 2 H), 3.70 (s, 3 H); HRMS (ESI) calcd. for
C22H18O4N2Cl (M+H)+ 409.0949, found: 409.0963.
N-(3-(Benzo[d]oxazol-2-yl)phenyl)-2-(4-methoxyphenoxy)acetamide (22). 35 mg, 89% yield;
1
1H NMR (400 MHz, DMSO-d6) δ H NMR (400 MHz, DMSO-d6) δ ppm 10.36 (s, 1 H), 8.67 (t, J
= 1.8 Hz, 1 H), 7.93 (d, J = 7.7 Hz, 1 H), 7.85 (br s, 1 H), 7.79 - 7.88 (m, 2 H), 7.58 (t, J = 8.0
18

Hz, 1 H), 7.41 - 7.46 (m, 2 H), 6.88 - 7.01 (m, 4 H), 4.69 (s, 2 H); HRMS (ESI) calcd. for
C22H19O4N2 (M+H)+ 375.1339, found: 375.1353.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-((4-methoxyphenyl)thio)acetamide (24). 24
mg, 60% yield; 1H NMR (400 MHz, DMSO-d6) δ ppm 10.49 (s, 1 H), 8.12 - 8.17 (m, 2 H), 7.77 -
7.85 (m, 4 H), 7.39 - 7.47 (m, 3 H), 6.90 - 6.96 (m, 2 H), 3.73 - 3.76 (m, 5 H); HRMS (ESI) calcd.
for C22H18O3N2ClS (M-H)- 423.0576, found: 423.0565.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-((4-methoxyphenyl)amino)acetamide (25). 11
mg, 29% yield; 1H NMR (300 MHz, DMSO-d6) δ ppm 10.60 (s, 1 H), 8.16 - 8.22 (m, 3 H), 7.80 -
7.95 (m, 4 H), 7.44 - 7.46 (m, 2 H), 6.89 - 7.06 (m, 2 H), 4.38 (s, 1 H), 4.09 (s, 2 H), 3.70 (s, 3
H); HRMS (ESI) calcd. for C22H19O3N3Cl (M+H)+ 408.1109, found: 408.1090.
N-(6-(5-Chlorobenzo[d]oxazol-2-yl)naphthalen-2-yl)-2-phenoxyacetamide (26). 32 mg, 87%
yield; 1H NMR (300 MHz, DMSO-d6) δ 10.48 (s, 1 H), 8.76 (s, 1 H), 8.45 (s, 1 H), 8.10 - 8.28 (m,
2 H), 8.04 (d, J = 8.7 Hz, 1 H), 7.94 (d, J = 2.1 Hz, 1 H), 7.69 - 7.90 (m, 2 H), 7.49 (dd, J = 8.6,
2.1 Hz, 1 H), 7.34 (dd, J = 8.6, 7.1 Hz, 2 H), 6.93 - 7.10 (m, 3 H), 4.79 (s, 2 H); HRMS (ESI)
calcd. for C25H18O3N2Cl (M+H)+ 429.1000, found: 429.1012.
N-(5-(5-Chlorobenzo[d]oxazol-2-yl)benzo[d]thiazol-2-yl)-2-(4-methoxyphenoxy)acetamide
1
(27). 29 mg, 78% yield; H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1 H), 8.90 (d, J = 1.6 Hz, 1
H), 8.25 (dd, J = 8.4, 1.6 Hz, 1 H), 7.91 - 7.96 (m, 3 H), 7.79 - 7.84 (m, 2 H), 7.48 - 7.78 (m, 2H),
6.92 (m, 1 H), 4.51 (s, 2 H); HRMS (ESI) calcd. for C23H17O4N3ClS (M+H)+ 466.0623, found:
466.0615.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(3-methoxyphenoxy)acetamide (30). 23 mg,
1
60% yield; H NMR (300 MHz, DMSO-d6) δ 10.46 (s, 1 H), 8.17 (br d, J = 8.4 Hz, 2 H), 7.86 -
7.95 (m, 3 H), 7.81 (d, J = 8.6 Hz, 1 H), 7.45 (dd, J = 8.6, 2.1 Hz, 1 H), 7.22 (t, J = 8.4 Hz, 1 H),
19

6.53 - 6.65 (m, 3 H), 4.75 (s, 2 H), 3.74 (s, 3 H); 13C NMR (75 MHz, DMSO-d6) δ 167.11,
163.63, 160.44, 158.93, 148.96, 142.94, 142.00, 130.01, 128.94, 128.44, 125.16, 120.79,
119.76, 119.23, 112.11, 106.93, 106.72, 101.15, 67.23, 55.12; HRMS (ESI) calcd. for
C20H14O3N2Cl (M+H)+ 407.0793, found: 407.0790.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(4-cyanophenoxy)acetamide (31). 21 mg,
53% yield; 1H NMR (300 MHz, DMSO-d6) δ 10.56 (s, 1 H), 8.17 (d, J = 8.7 Hz, 2 H), 7.89 - 7.95
(m, 6 H), 7.44 (dd, J = 8.7, 2.1 Hz, 1 H), 7.18 (d, J = 8.7 Hz, 2 H), 4.91 (s, 2 H); HRMS (ESI)
calcd. for C22H15O3N3Cl (M+H)+ 404.0797, found: 404.0803.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(4-fluorophenoxy)acetamide (32). 37 mg, 94%
yield; 1H NMR (300 MHz, DMSO-d6) δ 10.47 (s, 1 H), 8.17 (d, J = 8.6 Hz, 2 H), 7.86 - 7.95 (m, 3
H), 7.81 (d, J = 8.6 Hz, 1 H), 7.45 (dd, J = 8.6, 2.1 Hz, 1 H), 7.11 - 7.20 (m, 2 H), 7.00 - 7.09 (m,
2 H), 4.75 (s, 2 H); HRMS (ESI) calcd. for C21H15O3N2ClF (M+H)+ 397.0750, found: 397.0769.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(4-nitrophenoxy)acetamide (33). 10 mg, 23%
yield; 1H NMR (300 MHz, DMSO-d6) δ 10.43 (s, 1 H), 8.13 - 8.20 (m, 3 H), 7.88 - 7.99 (m, 4 H),
7.79 - 7.82 (m, 2 H), 7.25 - 7.49 (m, 2 H), 4.36 (s, 2 H); HRMS (ESI) calcd. for C21H15O5N3Cl
(M+H)+ 424.0695, found: 424.0674.
2-([1,1'-Biphenyl]-4-yloxy)-N-(4-(5-chlorobenzo[d]oxazol-2-yl)phenyl)acetamide (34). 27
mg, 61% yield; 1H NMR (400 MHz, DMSO-d6) δ ppm 10.54 (s, 1 H), 8.16 - 8.20 (m, 1 H), 7.79 -
8.00 (m, 5 H), 7.61 - 7.66 (m, 1 H), 7.39 - 7.51 (m, 3 H), 7.26 - 7.38 (m, 1 H), 7.11 (d, J = 8.8 Hz,
2 H), 4.83 (s, 1 H); HRMS (ESI) calcd. for C27H20O3N2Cl (M+H)+ 455.1157, found: 455.1153.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(4-isopropyl-3-methylphenoxy)acetamide
(35). 41 mg, 96% yield; 1H NMR (400 MHz, DMSO-d6) δ ppm 10.45 (s, 1 H), 8.17 (d, J = 8.8 Hz,
2 H), 7.79 - 7.98 (m, 4 H), 7.45 (dd, J = 8.7, 2.1 Hz, 1 H), 7.16 (d, J = 9.3 Hz, 1 H), 6.77 - 6.83
20

(m, 2 H), 4.71 (s, 2 H), 3.03 (sep, J = 6.9 Hz, 1 H), 2.27 (s, 3 H), 1.14 (d, J = 6.9 Hz, 6 H);
HRMS (ESI) calcd. for C25H24O3N2Cl (M+H)+ 435.1470, found: 435.1462.
N-(4-(5-Fluorobenzo[d]oxazol-2-yl)phenyl)-2-(4-methoxyphenoxy)acetamide (36). 37 mg,
96% yield; 1H NMR (300 MHz, DMSO-d6) δ 10.43 (s, 1 H), 8.16 (d, J = 8.6 Hz, 2 H), 7.91 (d, J =
8.6 Hz, 2 H), 7.80 (dd, J = 8.9, 4.4 Hz, 1 H), 7.66 (dd, J = 8.8, 2.6 Hz, 1 H), 7.27 (td, J = 9.3, 2.6
Hz, 1 H), 6.85 - 7.01 (m, 4 H), 4.69 (s, 2 H), 3.70 (s, 3 H); HRMS (ESI) calcd. for C22H18O4N2F
(M+H)+ 393.1245, found: 393.1264.
N-(4-(5-Fluorobenzo[d]oxazol-2-yl)phenyl)-2-phenoxyacetamide (37). 39 mg, quantitative
yield; 1H NMR (300 MHz, DMSO-d6) δ 10.48 (s, 1 H), 8.17 (d, J = 8.5 Hz, 2 H), 7.91 (d, J = 8.5
Hz, 2 H), 7.80 (dd, J = 8.9, 4.4 Hz, 1 H), 7.66 (dd, J = 8.8, 2.6 Hz, 1 H), 7.22 - 7.39 (m, 3 H),
6.95 - 7.07 (m, 3 H), 4.76 (s, 2 H); HRMS (ESI) calcd. for C21H16O3N2F (M+H)+ 363.1140, found:
363.1149.
N-(4-(5-Bromobenzo[d]oxazol-2-yl)phenyl)-2-(4-methoxyphenoxy)acetamide (38). 31 mg,
81% yield; 1H NMR (300 MHz, DMSO-d6) δ 10.43 (s, 1 H), 8.17 (d, J = 8.5 Hz, 2 H), 8.02 (d, J =
1.9 Hz, 1 H), 7.91 (d, J = 8.6 Hz, 2 H), 7.76 (d, J = 8.6 Hz, 1 H), 7.57 (dd, J = 8.6, 2.0 Hz, 1 H),
6.86 - 7.00 (m, 4 H), 4.69 (s, 2 H), 3.70 (s, 3 H); 13C NMR (75 MHz, DMSO-d6) δ 167.44,
163.42, 153.87, 151.74, 149.33, 143.42, 142.04, 128.44, 127.87, 122.14, 120.73, 119.76,
116.68, 115.72, 114.60, 112.60, 67.93, 55.35; HRMS (ESI) calcd. for C22H16O4N2Br (M+H)+
451.0288, found: 451.0307.
2-(4-Methoxyphenoxy)-N-(4-(5-phenylbenzo[d]oxazol-2-yl)phenyl)acetamide (39). 28 mg,
70% yield; 1H NMR (300 MHz, DMSO-d6) δ 10.40 - 10.46 (m, 1 H), 8.20 (br d, J = 8.5 Hz, 2 H),
8.03 (d, J = 1.7 Hz, 1 H), 7.89 - 7.97 (m, 2 H), 7.84 (d, J = 8.6 Hz, 1 H), 7.67 - 7.77 (m, 3 H),
7.46 - 7.54 (m, 2 H), 7.35 - 7.42 (m, 1 H), 6.86 - 7.01 (m, 4 H), 4.69 (s, 2 H), 3.70 (s, 3 H); 13C
NMR (75 MHz, DMSO-d6) δ 167.40, 153.88, 151.76, 149.77, 142.40, 141.76, 140.02, 137.54,
21

128.94, 128.24, 127.34, 127.08, 124.34, 119.79, 117.53, 115.72, 114.61, 110.96, 67.95, 55.35,
40.22; HRMS (ESI) calcd. for C22H16O4N2Cl (M-H)+ 407.0793, found: 407.0790.
2-(4-Methoxyphenoxy)-N-(4-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide (40). 29 mg, 74%
yield; 1H NMR (300 MHz, DMSO-d6) δ 10.48 (s, 1 H), 8.54 (dd, J = 4.8, 1.4 Hz, 1 H), 8.20 - 8.28
(m, 3 H), 7.95 (d, J = 8.6 Hz, 2 H), 7.45 (dd, J = 8.1, 4.8 Hz, 1 H), 6.87 - 7.02 (m, 4 H), 4.71 (s, 2
H), 3.72 (s, 3 H); HRMS (ESI) calcd. for C21H18O4N3 (M+H)+ 376.1292, found: 376.1297.
Phenyl (4-(5-chlorobenzo[d]oxazol-2-yl)phenyl)carbamate (19). To a solution of 4-(5-
chlorobenzo[d]oxazol-2-yl)aniline (50 mg, 1 eq) in anhydrous DCM (5 mL) at 0 °C, phenyl
chloroformate (31 µL, 1.2 eq) and triethylamine (42 µL, 1.5 eq) was added. The reaction mixture
was warmed to room temperature and stirred for 3 h. The reaction was quenched by water (10
mL) and extracted with multiple portions of ethyl acetate (3 × 5 mL). The combined organic
extracts were combined, washed with saturated solution of sodium bicarbonate (10 mL) and
brine (10 mL), dried over sodium sulfate (10 g), filtered and concentrated. Compound 19 was
purified by silica gel column chromatography in 50% ethyl acetate/hexanes to yield a white solid
1
(62 mg, 83% yield). H NMR (400 MHz, DMSO-d6) δ ppm 10.72 (s, 1 H), 8.15 - 8.20 (m, 2 H),
7.89 (d, J = 2.1 Hz, 1 H), 7.81 (d, J = 8.6 Hz, 1 H), 7.74 - 7.78 (m, 2 H), 7.42 - 7.48 (m, 3 H),
7.25 - 7.34 (m, 3 H); HRMS (ESI) calcd. for C21H16ClO4N2 (M+H)+ 365.0688, found: 365.0694.
4-Methoxyphenyl (4-(5-chlorobenzo[d]oxazol-2-yl)phenyl)carbamate (23). To a solution of
4-(5-chlorobenzo[d]oxazol-2-yl)aniline (50 mg, 1 eq) in anhydrous DCM (5 mL) at 0 °C, 4-
methoxyphenyl chloroformate (36 µL, 1.2 eq) and triethylamine (42 µL, 1.5 eq) was added. The
reaction mixture was warmed to room temperature and stirred for 3 h. The reaction was
quenched by water (10 mL) and extracted with multiple portions of ethyl acetate (3 × 5 mL). The
combined organic extracts were combined, washed with saturated solution of sodium
bicarbonate (10 mL) and brine (10 mL), dried over sodium sulfate (10 g), filtered and
22

concentrated. Compound 23 was purified by silica gel column chromatography in 50% ethyl
1
acetate/hexanes to yield a white solid (81 mg, 79% yield). H NMR (400 MHz, DMSO-d6) δ
10.66 (s, 1 H), 8.16 (s, 1 H), 7.89 (d, J = 2.1 Hz, 1 H), 7.82 (d, J = 8.6 Hz, 1 H), 7.73 - 7.78 (m, 2
H), 7.45 (dd, J = 8.6, 2.2 Hz, 1 H), 7.16 - 7.22 (m, 2 H), 6.96 - 7.01 (m, 2 H), 3.78 (s, 3 H);
HRMS (ESI) calcd. for C21H16ClO4N2 (M+H)+ 395.0793, found: 395.0792.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(4-methoxyphenoxy)-N-methylacetamide
(28). To a solution of 12 (10 mg, 1.0 eq) in anhydrous THF (1 mL) at 0 °C, LiHMDS (30 μL, 1.2
eq, 1 M in THF) was added the suspension was stirred for 30 min. Methyl iodide (5 µL, 3.3 eq)
was added to this solution at 0 °C and the reaction mixture was warmed to room temperature
and stirred overnight. Upon complete consumption of 12, the reaction mixture was
concentrated. Compound 28 was purified by silica gel column chromatography in 30% ethyl
acetate/hexanes to yield a white solid (8.9 mg, 86% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.23
(d, J = 8.4 Hz, 2 H), 7.94 (d, J = 2.0 Hz, 1 H), 7.84 (d, J = 8.4 Hz, 1 H), 7.66 (d, J = 8.4 Hz, 2 H),
7.48 (dd, J = 8.7, 2.4 Hz, 1 H), 6.76 - 6.82 (m, 4 H), 4.63 (br s, 3 H), 3.66 (s, 3 H), 3.30 (s, 3 H);
HRMS (ESI) calcd. for C23H20O4N2Cl (M+H)+ 423.1106, found: 423.1110.
N-(4-(5-Chlorobenzo[d]oxazol-2-yl)phenyl)-2-(4-methoxyphenoxy)-N-(prop-2-yn-1-
yl)acetamide (29). To a solution of 12 (50 mg, 1.0 eq) in anhydrous DMF (3 mL) at 0 °C,
sodium hydride (6 mg, 60% suspension in mineral oil, 1.2 eq) was added the suspension was
stirred for 15 min. Propargyl bromide (22 µL, 1.2 eq, 80 wt% solution in toluene) was added
dropwise to this solution at 0 °C and he reaction mixture was warmed to room temperature and
stirred overnight. Upon complete consumption of 12, the reaction mixture was quenched by ice-
water mixture (5 mL) and extracted with multiple portions of ethyl acetate (3 × 5 mL). The
combined organic extracts were combined, washed with brine (10 mL), dried over sodium
sulfate (10 g), filtered and concentrated. Compound 29 was purified by silica gel column
1
chromatography in 30% ethyl acetate/hexanes to yield a yellow solid (24 mg, 44% yield). H
23

NMR (300 MHz, DMSO-d6) δ 8.07 (d, J = 8.9 Hz, 2 H), 7.82 (d, J = 2.1 Hz, 1 H), 7.76 (d, J = 8.7
Hz, 1 H), 7.39 (dd, J = 8.6, 2.2 Hz, 1 H), 7.08 (d, J = 9.1 Hz, 2 H), 6.84 - 6.90 (m, 4 H), 4.31 (d, J
= 2.3 Hz, 3 H), 3.57 - 3.65 (m, 1 H), 3.23 (t, J = 2.1 Hz, 2 H); HRMS (ESI) calcd. for
C25H20O4N2Cl (M+H)+ 447.1106, found: 447.1105.
ASSOCIATED CONTENT
Biological protocols and Supplementary Figures (S1-S3)
Synthetic schemes and analytical data for compounds 12, 30, 38, and 39.
AUTHOR INFORMATION
Corresponding Author
deiters@pitt.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
A.D. acknowledges support by a Research Scholar Grant (120130-RSG-11-066-01-RMC) from
the American Cancer Society. Y.N. was supported, in part, by an Andrew Mellon Predoctoral
Fellowship. We thank Dr. Bogi Andersen (University of California Irvine) for supplying the miR-
21 promoter reporter plasmid.
24

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